2,4-disulfonyl phenyl butyl nitrone, its salts, and their use as pharmaceutical free radical traps

ABSTRACT

2,4-disulfonyl α-phenyl-tert-butyl nitrone and its pharmaceutically acceptable salts are disclosed. These materials are useful as pharmaceutical agents for oral or intravenous administration to patients suffering from acute central nervous system oxidation as occurs in a stroke or from gradual central nervous system oxidation which can exhibit itself as progressive central nervous system function loss.

BACKGROUND OF THE INVENTION

This invention relates to a particular nitrone compound and its saltsand their advantageous use as pharmaceutical nitrone free radicaltrapping agents.

BACKGROUND INFORMATION

Alpha-phenyl tert butyl nitrone ##STR1## or "PBN") was identified in the1970s as a useful analytical reagent to be used in conjunction withelectron spin resonance ("ESR") to aid in the detection of freeradicals. PBN was found to react with certain free radicals and generatea chemical species yielding a characteristic ESR spectrum and thusmaking it possible to determine the presence or absence of freeradicals.

In the late 1970s and early 1980s the medical community began to focuson the roles played by free radicals in diseases such as heart attacks,strokes and the like. PBN was used increasingly in vitro to provideanalytical evidence of the presence of free radicals in these settings.It was also later administered in vivo in animal models, again as ananalytical adjunct in attempts to observe free radicals during ischemiasimulations and the like.

In the mid 1980s, the first possible therapeutic effects of PBN wereimplied when severe trauma ischemia animal tests showed that PBN-treatedanimals were more likely to survive than controls.

On May 2, 1991, our PCT patent application WO-91-05552 was published.This patent application, which in part corresponds to now-issued U.S.Pat. Nos. 5,025,032 and 5,036,097, described PBN and a family of PBNderivatives defined by the formula ##STR2## X is phenyl or ##STR3##where R is H, ##STR4## or Z; or ##STR5## and n is a whole integer from 1to 5 or ##STR6## and Y is tert-butyl or a hydroxylated or acetylatedtert-butyl or a substituted phenyl. These compounds were proposed aspharmaceutical agents to treat the aftermath of stroke and otherconditions reported to be associated with free radical damage.

In 1992 we filed a second PCT patent application on PBN and relatedcompounds and their medical use. This application, based on prior U.S.patent application Ser. No. 716,952 (filed Jun. 18, 1991) and abandonedon Apr. 26, 1993, was published on Dec. 23, 1992 as WO 92/22290. This1992 publication provided two extremely broad and general disclosures.First, it attempted to describe as many disease states as possible whichwere associated with free radicals. These ranged from CNS conditions(including stroke, aging, migraine, etc.) through peripheral organdisease (including atherosclerosis, bed sores, wounds, and muscleoverexertion) through UV exposure, to mention but a few highlights.Second, it attempted to list as many potential spin traps as possible.

In addition to a whole range of non-PBN materials, this applicationgreatly expanded the definition of potentially useful PBN compounds toinclude PBN, and derivatives thereof of the formula ##STR7## wherein Xis phenyl, imidazolyl, phenothiazinyl or ##STR8## n=1-5, preferably 1-3;R² =independently (can vary within the molecule) halogen, alkyl,oxyalkyl, alkenyl, oxyalkenyl, OH, NH₂, NHZ, NZ₂, NO, ##STR9## --SO₃ H,--OSO₃ H, SH, --S(alkyl), --S(alkenyl, and haloalkyl, specificallyincluding --CF₃ ;

A=O or S; and

Z is a C₁ to C₆ straight, branched, alkyl or cyclic group; and

Y is a tert-butyl group that can be hydroxylated or acetylated at one ormore positions; phenyl or ##STR10##

PBN was stated to be the most preferred compound at that time, beingsaid to have no measurable effect on normal or uninjured cells, and anumber of derivatives were also stated to be useful, including hydroxyderivatives, especially 2-, 3- or 4-hydroxyphenyl t-butyl nitrone andphenyl (mono-, di- or trihydroxy) tert-butyl nitrone; PBN esters,especially esters which release 2-, 3-, or 4-hydroxyphenyl t-butylnitrone such as acetoxy derivative; 2-, 3-, or 4-carboxyphenyl t-butylnitrone; phenyl hydroxybutyl nitrone; alkoxyl derivatives, especiallyalkoxyl derivatives which release 2-, 3-, or 4-hydroxyphenyl t-butylnitrone, for example, the 2-, 3-, or 4-methoxyphenyl derivatives of PBN;and acetamide derivatives, especially acetamide derivatives whichrelease 2-, 3-, or 4-aminophenyl t-butyl nitrone; diphenyl nitrone (PPN)and the analogous diphenyl nitrone derivatives;N-tert-butyl-α-(4-nitro-phenyl) nitrone; andN-tert-butyl-α-(2-sulfophenyl) nitrone.

STATEMENT OF THE INVENTION

We have now discovered that one particular PBN derivative and its saltshave unexpectedly superior pharmacological properties. Although thisderivative, 2,4-disulfonyl PBN, falls within the broad family ofmaterials generally described in the aforementioned WO 92/022290publication, it is not specifically disclosed. Neither are itsadvantageous properties predicted.

The present compound with its two sulfonate groups was expected toexhibit improved water solubility but was also expected to exhibit poortransport across the blood/brain barrier because of its lipophobiccharacter. However, when the present compound was made and tested invivo, it showed an unexpected increase in efficacy as compared to PBN.This increase in efficacy occurred along with an increase in potency ascompared to PBN. In direct contrast to this marked increase in potencyand efficacy there was a marked and highly significant decrease intoxicity as compared to PBN.

These results were unexpected because in the general literature onstructure/activity relationships within specific defined families ofcompounds therapeutic potency typically covaries with toxicity. Thus,most related compounds maintain their ratio of therapeutic potency totoxicity. In contrast, the compound of this invention deviates from thisexpected relationship when its potency increased and its toxicitydecreased relative to closely related analogs.

Accordingly, in one aspect, the invention provides the PBN-disulfonylcompound ##STR11## and its pharmaceutically acceptable salts.

In a second aspect, the invention provides intravenously- andorally-administrable pharmaceutical compositions having this compound orits salt as active ingredient.

In a third aspect, this invention provides a method for treating apatient who is suffering from a condition involving acute oxidativedamage to the central nervous system, such as a patient who has suffereda stroke, in which a pharmaceutical composition based on this compoundor its salt is administered intravenously.

In a fourth aspect, this invention provides a method for treating apatient suffering from a condition characterized by protracted low gradeoxidative stress upon the central nervous system and progressive loss ofcentral nervous system function. In this method, a pharmaceuticalcomposition based on this compound or its salt is administeredintravenously or preferably orally.

DETAILED DESCRIPTION OF THE INVENTION

This Detailed Description is arranged into the following sections:

Brief Description of Drawings.

The Compounds and Salts.

Compound Preparation.

Pharmaceutical Compositions.

Conditions Treated and Treatment Regimens.

Examples.

BRIEF DESCRIPTION OF THE DRAWINGS

In this specification reference will be made to the accompanyingdrawings in which

FIG. 1 is a schematic flow chart of the reactions used to prepare thecompound.

FIGS. 2 (A, B and C) and 3 (A, B, C, and D) are two sets of graphsillustrating the undesirable change in animal body thermal regulatoryability which occurs as a function of dose level with a prior artnitrone radical trapping agent and contrasting this with the lack ofsuch undesired toxic effect with the compound of the invention.

FIG. 4 (A, B, C, and D) is a set of four graphs demonstrating thesuperiority of the compound of the invention as compared to a closelyrelated prior art nitrone compound in the treatment of gradualneurodegeneration conditions (such as Alzheimer's disease) asillustrated by their relative ability to interfere with beta amyloidprotein's inactivation of key enzymes in solution.

The Compound and Salts

The compound of this invention is 2,4-disulfonyl α-phenyl tertiary butylnitrone. It is also referred to informally herein as "2 4-disulfonylPBN" or "PBN 2,4-disulfonate." It exists in an acid form ##STR12## as asolid and in solution in low pH conditions. It also exists at higher pHsin an ionized salt form which can be shown as ##STR13## where X is apharmaceutically acceptable cation. Most commonly, this cation is amonovalent material such as sodium, potassium or ammonium, but it canalso be a multivalent alone or cation in combination with apharmaceutically acceptable monovalent anion, for example calcium with achloride, bromide, iodide, hydroxyl, nitrate, sulfonate, acetate,tartrate, oxalate, succinate, palmoate or the like anion; magnesium withsuch anions; zinc with such anions or the like. When these combinationsof a polyvalent cation and a monovalent anion are illustrated instructural formulae, herein, the monovalent anion is identified as "Y".

Among these materials, the free acid and the simple sodium, potassium orammonium salts are most preferred with the calcium and magnesium saltsalso being preferred but somewhat less so.

Compound Preparation

As detailed in FIG. 1 and demonstrated in Example 1, the compound ofthis invention can be prepared by a two step reaction sequence. In thefirst step, commercially available tertiary butyl nitrate(2-methyl-2-nitropropane) is converted to the corresponding n-hydroxylamine using a suitable catalyst such as an activated zinc/acetic acidcatalyst or an aluminum/mercury amalgam catalyst. This reaction can becarried out in 0.5 to 12 hours and especially about 2 to 6 hours or soat a temperature of about 15° to 100° C. in a liquid reaction mediumsuch as alcohol/water mixture in the case of the zinc catalyst or anether/water mixture in the case of the aluminum amalgam catalyst.

In the second step, the freshly formed hydroxylamine is reacted with4-formyl-1,3-benzenedisulfonic acid, typically with a slight excess ofthe amine being used. This reaction can be carried out at similartemperature conditions. This reaction is generally complete in 10 to 24hours.

The product so formed is the free acid and is characterized by amolecular weight of 89 g/mole. It is a white powdery material whichdecomposes upon heating. It is characterized by a solubility in water ofgreater than 1 gram/ml and a ¹ H NMR spectrum in D₂ O of 8.048 ppm (dd,8.4, 1.7 Hz); 8.836 ppm (d, 8.4 Hz); 8.839 ppm (d, 1.7 Hz); 8.774 ppm(s).

The various salts can be easily formed by admixing the free acid inaqueous medium with two equivalents of the appropriate base, forexample, KOH for the potassium salt, and the like.

Pharmaceutical Compositions

The compound (including its salts) can be formulated into pharmaceuticalcompositions suitable for oral or intravenous injection administration

The compositions for oral administration can take the form of liquidsolutions or suspensions, powders, tablets, capsules or the like. Insuch compositions, the PBN 2,4-disulfonate or its salt is usually aminor component (0.1 to say 50% by weight) with the remainder beingvarious vehicles or carriers and processing aids helpful for forming thedesired dosing form. A liquid form may include a suitable aqueous ornonaqueous vehicle with buffers, suspending dispensing agents,colorants, flavors and the like.

A solid form may include, for example, any of the following ingredients,or compounds of a similar nature: a binder such as microcrystallinecellulose, gum tragacanth or gelatin; an excipient such as starch orlactose, a disintegrating agent such as alginic acid, Primogel, or cornstarch; a lubricant such as magnesium stearate; a glidant such ascolloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; or a flavoring agent such as peppermint, sugar, methylsalicylate, or orange flavoring.

In the case of injectable compositions, they are commonly based uponinjectable sterile saline or phosphate-buffered saline or otherinjectable carriers known in the art. Again the active nitrone istypically a minor component, often being from about 0.05 to 10% byweight with the remainder being the injectable carrier and the like.

Conditions Treated and Treatment Regimens

The conditions treated with the 2,4-disulfonyl PBN generally fall intotwo groups. The first includes conditions involving acute intenseoxidative damage to a region of the central nervous system. Examples ofthese conditions include stroke, conditions associated with stroke,concussion and subarachnoid hemorrhage. In this setting, the compound isadministered in manners designed to get the drug into the patient'sbloodstream as quickly and directly as possible. This usually meansintravenous administration.

Intravenous dose levels for treating these conditions range from about0.1 mg/kg/hour to at least 10 mg/kg/hour, all for from about 1 to about120 hours and especially 24 to 96 hours. A preloading bolus of fromabout 10 to about 500 mg may also be administered to achieve adequatesteady state levels.

An unexpected and key advantage of 2,4-disulfonyl PBN is that it can beadministered at vastly higher levels than are possible with PBN itself.As will be shown in the Examples, doses of up to 1000 mg/kg/hour andhigher or intravenous bolus doses of from 10 to 2500 mg/kg have beendemonstrated to be possible with PBN-2,4-disulfonyl or its salts whilewith PBN itself death or acute toxicity results from such doses. With2,4 disulfonyl PBN there is an unexpected positive continuance of thedose/response curve in these high dose levels with the clear messagethat intense heavy dosing immediately post stroke or other trauma may inmany cases provide a major positive impact upon recovery.

The second group of conditions which respond favorably to 2,4-disulfonylPBN treatment are conditions characterized by protracted low gradeoxidative stress upon the central nervous system and gradual progressivecentral nervous system function loss. These conditions includeAlzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis(ALS), multi-infarct dementia, retinopathy and the like. Each of theseconditions is characterized by a progressive loss of function.2,4-disulfonyl-PBN or its salts, when administered orally orintravenously, can slow and possibly reverse the loss of function. Ifintravenous administration is desired, similar levels to those used withacute conditions but at the lower end of the ranges are generally used.

In these cases, the regimen for treatment may stretch over many monthsor years so oral dosing is preferred for patient convenience andtolerance. With oral dosing, one to three oral doses per day, each fromabout 0.02 to about 50 mg/kg are called for with preferred doses beingfrom about 0.04 to about 5.0 mg/kg.

Of course, one can administer 2,4-disulfonyl PBN as the sole activeagent or one can administer it in combination with other agents.

EXAMPLES EXAMPLE 1

Synthesis of 2,4-disulfonylphenyl-N-t-butylnitrone (Compound "I" insubsequent Examples). This preferred synthesis is based on the work byR. H. Hinton and E. G. Janzen (J. Org. Chem. 57:2646-2651, 1992). Asshown in FIG. 1 it involves the condensation of an aldehyde with ahydroxylamine. The hydroxylamine is unstable and is prepared fresh onthe day of use using an activated zinc catalyst. The synthesis is asfollows:

Prerequisite Chemicals

1. 95% Ethanol

2. 2-Methyl-2-nitropropane

3. Zinc dust

4. Glacial acetic acid

5. Diethyl ether

6. Saturated sodium chloride

7. Magnesium Sulfate, Anhydrous solid

8. 4-Formyl-1,3-benzenesulfonic acid (MW 310.21 g/mole), disodium salt,hydrate

9. Methanol

10. Dichloromethane

Procedure

Preparation of N-t-Butylhydroxylamine

1. A 500 mL three neck round bottom flask is equipped with a magneticstir bar, thermometer adapter, thermometer, and addition funnel.

2. 95% ethanol (350 mL) was added to the flask and cooled to 10° C. inan ice bath.

3. 2-Methyl-2-nitropropane (6.18 g, 0,060 mole), and zinc dust (5.89 g,0,090 mole) were added in single portions.

4. Glacial acetic acid (10.8 g, 0,180 mole) was placed in the additionfunnel and added dropwise at such a rate with vigorous stirring tomaintain the temperature below 15° C.

5. The ice bath was removed and mixture was stirred for 3 hrs at roomtemperature.

6. The solvent was stripped from the mixture, leavingt-butylhydroxylamine, zinc acetate, and water.

7. Dichloromethane (50 mL) was added and the mixture filtered through aBuchner funnel.

8. The zinc acetate cake left on the filter paper was washed with 2×25mL dichloromethane.

9. Water was separated from the filtrate in a separatory funnel and theorganic layer dried over magnesium sulfate.

10. The magnesium sulfate was removed by filtering through fluted filterpaper, then dichloromethane stripped off by rotary evaporation.

11. The product (100% yield=5.34 g), a viscous liquid, was dissolved inmethanol (50 mL) for use in part B.

Preparation of 2,4-disulfonylphenyl-N-t-butylnitrone

1. A 3-neck 250 ml round bottom flask was set up with a stir bar, a gasdispersion tube, an addition funnel, and a Friedrichs condenser cooledwith recirculating ice water.

2. To the flask were added 200 mL of methanol,4-formyl-1,3-benzenedisulfonic acid (9.31 g, 30 mmoles) andN-t-butylhydroxylamine (25 mL of the methanol solution from part A, 30mmoles theoretical).

3. The reaction was heated to reflux with a heating mantle whilebubbling the reaction with nitrogen with stirring.

4. The mixture was refluxed for 2 hours.

5. The remainder of hydroxylamine from part A was added.

6. Refluxing was continued with nitrogen bubbling for at least 18 hours,but not more than 24 hours.

7. The hot reaction mixture was filtered on a Buchner funnel, and thesolid washed with hot methanol.

8. The methanol was stripped off by rotary evaporation to a yellow,viscous oil.

9. Hot 1:1 ethanol:acetone (200 mL) was added and the mixture heated todissolve the oil.

10. The solution was cooled to crystallize the product.

11. The product was collected on a Buchner funnel and dried under vacuumovernight.

12. The reaction typically gives 75% yield of I, a white powder.

EXAMPLE 2

Alternate Synthesis of 2,4-disulfonylphenyl-N-t-butylnitrone (CompoundI). This is an earlier-developed method which used to prepare samples ofthe compound used in several of the experiments reported in the Examplesof this specification. The product of this Example is identical in allways to the product of Example 1. This synthesis method is as follows:

Prerequisite Chemicals

1. Aluminum Foil, cut into 5 cm wide strips and rolled in a ca. 1 cmdiameter cylinder

2. Mercury (II) Chloride (9.68 g in 476 ml water)

3. Ethanol

4. Ether (6 L)

5. Pure Water

6. 2-Methyl-2-nitropropane

7. Sodium Hydroxide, 2M (80 g in 1 L water)

8. Magnesium Sulfate, Anhydrous solid

9. 4-Formyl-1,3-benzenesulfonic acid (MW 310.21 g/mole)

Procedure

Preparation of N-t-Butylhydroxylamine

1. Aluminum foil cylinders were dipped into HgCl₂ solution for 15-30seconds, then dipped in ethanol, then dipped in ether and then placedinto a 5L flask containing 500 ml of diethyl ether and 21.4 ml of water.

2. The flask was fitted with a 250 ml pressure-equalizing droppingfunnel, a mechanical stirrer, a nitrogen inlet, and a Friedrichscondenser cooled with recirculated ice water.

3. The mixture was stirred for 10 minutes.

4. 2-Methyl-2-nitropropane (71.68 g, 75.5 ml) was added using thedropping funnel at such a rate as to maintain a vigorous reflux.

NOTE: Addition must be completed in less than 20 minutes or the yielddrops significantly.

5. As the addition proceeded, ether was added in 500 ml portions. Thiswas done to maintain as high a concentration of product as possiblewithout the formation of a gel. Up to 2 L of ether can be added with nodeleterious effects on the yield.

6. Once addition of 2-methyl-2-nitropropane was complete, the reactionwas stirred for an additional 30 minutes.

7. The resulting gray suspension was suction filtered in 3 batches toremove aluminum salts.

8. Each filter cake was washed with 1 L of ether.

9. The combined either layers were washed with 300 ml of 2 M NaOH, thendried (MgSO₄), and concentrated in vacuo to leave a soft white solid.

10. The solid melts just above room temperature, but could be driedfurther in a vacuum oven (no more than a few minutes), leaving 38 to 45g of solid.

11. The solid can be used as is or was purified by recrystallizationfrom pentane.

12. Molecular weight-89 g/mole.

B. Preparation of 2,4-disulfonylphenyl-N-t-butylnitrone

1. A 250 ml flask was equipped with a stir bar and a Friedrichscondenser cooled with recirculated ice water.

2. The flask was charged with 71.8 ml of methanol, 14.5 g of4-formyl-1,3-benzenedisulfonic acid (46.7 mmoles, 1 eq.), and 5.0 g ofN-t-butylhydroxylamine (56.2 mmoles, 1.2 eq.).

3. The mixture was refluxed overnight.

4. The reaction product was transferred to round-bottom flask androtovaped to dry.

5. The solid residue was mashed with ether, the ether was decanted off(yellow).

6. Step 5 was repeated.

7. Product ("I") was crystallized from methanol following a hot methanolfiltration to remove insoluble precipitates and recrystallized twicefrom methanol.

EXAMPLE 3

A series of experiments were carried out to compare in vivo the efficacyof 2,4 disulfonyl PBN ("I"), PBN, and two monosulfonate PBN compounds asagents for protecting against neuron loss following brain ischemia andreperfusion injury. The test procedure is that reported by W. Cao, J. M.Carney, A. Duchon, R. A. Floyd and M. Chevion as "Oxygen free radicalinvolvement in ischemia and reperfusion injury to brain, NeuroscienceLetters, 88 (1988), 233. In the experiments a test compound wasadministered to groups of six gerbils i.p. as a single dose 30 minbefore 5 min bilateral carotid occlusion. The density of neuronal nucleiin a 100 micron was measured. Two controls were present--controls whichreceived no test compound and controls which received no test compoundand no brain ischemia. As illustrated in Table 1. the compound of theinvention showed unexpected advantages as compared to the prior artmaterials. First, it was seen that at low dose levels, such as 3.2mg/kg, compound I was 2-3 times as potent at preventing neuronal loss.At high does levels it was seen that I was able to achieve completeprotection against neuronal loss as the test brains showed neuronaldensities identical to the non-ischemic controls. The prior materialswere either toxic at these dose levels or showed significantly lowerdegrees of protection. These results show a clear increase in potencyfor neural protection for compound I compared to PBN and two closelyrelated analogs and an unexpected decrease in toxicity compared to PBN.

                  TABLE 1                                                         ______________________________________                                                      Neuronal nuclei/100 micron field                                                    2-       3-                                                             PBN   sulfo    sulfo  I.                                        ______________________________________                                        Non-ischemic control                                                                          4.21    4.21     4.21 4.21                                                    (.43)   (.43)    (.43)                                                                              (.43)                                   Ischemic control                                                                              0.58    0.58     0.58 0.58                                                    (.28)   (.28)    (.28)                                                                              (.28)                                   3.2 mg/kg       0.43    0.73     0.35 1.43                                                    (.18)   (.34)    (.21)                                                                              (.31)                                   10 mg/kg        1.13    0.68     0.81 2.57                                                    (.39)   (.31)    (.40)                                                                              (.25)                                   32 mg/kg        1.83    0.73     1.63 3.53                                                    (.21)   (.31)    (.35)                                                                              (.41)                                   50 mg/kg        3.11    1.01     1.63 4.11                                                    (.29)   (.61)    (.35)                                                                              (.43)                                   100 mg/kg       3.68    0.93     1.93 4.18                                                    (.71)   (.53)    (.39)                                                                              (.49)                                   320 mg/kg       3.78    1.11     1.78 4.23                                                    (.43)   (.41)    (.40)                                                                              (.39)                                   1000 mg/kg      Toxic   0.98     1.58 4.11                                                            (.43)    (.38)                                                                              (.41)                                   3200 mg/kg      Toxic   --       --   4.18                                    ______________________________________                                    

EXAMPLE 4

A series of experiments were conducted in which compound I was comparedto PBN and two sulfonate analogs in post-ischemia treatment. The generalmethod described in example 1 was used but the test compounds wereadministered i.p. as a single dose 30 min after reperfusion following 5min ischemia. The results are summarized in Table 2. They show that thecompound of the invention is again more potent at low doses and morepotent and less toxic at high doses. Also again, toxicity interfereswith the prior art materials ability to go to high doses at which levelsthe compound of the invention provides dramatically effective therapy.

                  TABLE 2                                                         ______________________________________                                                      Neuronal nuclei/100 micron field                                                    2-       3-                                                             PBN   sulfo    sulfo  I.                                        ______________________________________                                        Non-ischemic control                                                                          4.18    4.18     4.18 4.18                                                    (.59)   (.59)    (.59)                                                                              (.59)                                   Ischemic control                                                                              0.85    0.85     0.85 0.85                                                    (.19)   (.19)    (.19)                                                                              (.19)                                   32 mg/kg        1.09    ND       ND   1.83                                                    (.31)                 (.41)                                   50 mg/kg        1.85    0.68     0.73 2.73                                                    (.49)   (.31)    (.34)                                                                              (.39)                                   100 mg/kg       2.11    0.78     1.09 3.41                                                    (.51)   (.23)    (.48)                                                                              (.37)                                   320 mg/kg       2.25    0.81     0.93 3.55                                                    (.43)   (.31)         (.48)                                   1000 mg/kg      Toxic   ND       ND   3.68                                                                          (.39)                                   ______________________________________                                    

EXAMPLE 5

Compound I was compared with PBN to determine their relativeeffectiveness for protection of neuronal loss when administered i.v. 60min after reperfusion onset following 5 min ischemia in gerbils usingthe general test method described in Example 1.. The results aresummarized in Table 3. and illustrate that compound I is ofsignificantly greater therapeutic benefit in a clinical treatmentsetting following injury to the brain.

                  TABLE 3                                                         ______________________________________                                                     mg/kg single dose                                                N = 6 per group                                                                              0.0    0.5       1.0  10                                       ______________________________________                                        Saline, no ischemia                                                                          4.11   --        --   --                                                      (.28)                                                          saline, ischemia                                                                             0.93   --        --   --                                                      (.17)                                                          PBN            --     0.83      1.07 1.23                                                           (.23)     (.29)                                                                              (.31)                                    I.             --     1.25      1.75 2.43                                                           (.19)     (.28)                                                                              (.31)                                    ______________________________________                                    

Neither PBN nor I. had an effect on neuronal density in control gerbilswithout brain injury.

EXAMPLE 6

Brain injury can manifest itself as behavioral changes. In thisexperiment, young adult (3-4 months of age) gerbils were tested todetermine their ability to perform an 8-arm maze test 24 hours followingan ischemic event as described in Example 1. As compared to nonischemicanimals, when untreated they committed many more errors. PBN andcompound I were administered to some of the test animals. As detailed inTable 4. gerbils treated with high doses of compound I had error levelsindistinguishable from those of nonischemic animals. PBN was lesseffective. This shows that compound I can protect against the loss oftemporal/spatial short term memory following ischemia (24 hours post)errors in 8-arm radial maze test of young gerbils following 5 minischemia.

                  TABLE 4                                                         ______________________________________                                                  mg/kg/hr for 24 hours                                               N = 6 per group                                                                           0.0      1.0     32     50   100                                  ______________________________________                                        Control     4.1      --      --          --                                               (.38)                                                             Postischemic                                                                              37.6     --      --          --                                               (4.85)                                                            PBN         --       29.8    18.19  6.23 5.83                                                      (7.27)  (5.83) (.71)                                                                              (.49)                                I.          --       14.63   7.19   4.28 4.11                                                      (3.81)  (.81)  (.29)                                                                              (.19)                                ______________________________________                                    

EXAMPLE 7

The ability of the compound of the invention to reduce infarct volumefollowing an ischemic event was determined. As detailed in Table 5. itwas observed that while PBN and compound I were both effective at lowdoses, at high doses I gave the best protection and PBN was toxic. Table5 shows the infarct volume observed when test compound was administeredi.v. 60 min after middle cerebral occlusion and continued for 24 hoursin C57BL/6J mice.

                  TABLE 5                                                         ______________________________________                                        Posttreatment  Infarct Volume in mm.sup.3                                     (mg/kg/hr)     0.0    1.0      10    100                                      ______________________________________                                        Control, no ischemia                                                                          0     --       --    --                                       Saline, ischemia                                                                             23     --       --    --                                                       (2)                                                           PBN            --     17.7     13.8  Toxic                                                          (2.8)    (2.3)                                          I.             --     16.8     12.7  8.3                                                            (1.7)    (3.93)                                                                              (.71)                                    ______________________________________                                    

EXAMPLE 8

In this study, compound I and PBN were compared for their ability toimpart lethality protection (% survived) in aged gerbils (18-24 monthsof age, n=12/group) from 10 min ischemia when given 30 min beforeischemia. As shown in Table 6. compound I was superior at all doselevels and achieved complete protection at high levels where PBN wasonly partially effective.

                  TABLE 6                                                         ______________________________________                                        Pretreatment (mg/kg)                                                                         0.0     10    32    100  320                                   ______________________________________                                        Saline         11      --    --    --   --                                    PBN            --      42    50     75   92                                   I.             --      50    75    100  100                                   ______________________________________                                    

EXAMPLE 9

An important advantage of the compound of this invention as compared tothe art-taught compound, PBN, is its markedly diminished toxicity. Asdetailed in Table 7. acute lethality in C57BL/6L mice was determinedbased upon varying sizes of single i.p. doses of nitrone. PBN showedsignificant toxicity at 560 mg/kg dose levels. Compound I showed notoxicity at doses nearly twenty times as great.

                  TABLE 7                                                         ______________________________________                                        % Survival mg/kg                                                              n = 20 mice                                                                              320      560    1000   3000 10000                                  ______________________________________                                        PBN        100       25     0      0    0                                     I.         100      100    100    100  100                                    ______________________________________                                    

EXAMPLE 10

Another undesirable systemic effect which has been observed in vivo withnitrone radical traps is a depression in body temperature. This toxicitycan have serious health consequences and also can complicate diagnosisof other conditions. As detailed in FIGS. 2 and 4, the compound of thisinvention was administered to mice and gerbils at levels as high as 1000mg/kg with no measurable temperature decrease. In contrast, the compoundof the art, PBN, gave up to an 8° C. decrease in body temperature at adoes of only 500 mg/kg.

EXAMPLE 11

The compound of the invention was tested to determine its effectivenessin the treatment of conditions characterized by protracted low gradeoxidative stress upon the central nervous system and gradual progressivecentral nervous system function loss by testing its effectiveness in amodel for Alzheimer's disease ("AD"). This model has the followingbasis: Recent studies have demonstrated that there is an age-associatedincrease in protein oxidation and loss of enzyme activities in the brainof aged individuals. Tissue cultures of fibroblasts from agedindividuals and red blood cells of different ages both show anexponential increase in protein carbonyl content (a measure of proteinoxidation) and a decrease in marker enzyme activities. Brain proteinoxidation progressively increases over the life span of the individual.

The role of abnormal amyloid precursor peptide processing and metabolismin AD has also been explored in a number of different models. In vitrostudies using embryonic hippocampal neuronal and neuronal/glial cultureshave demonstrated that βAP 1-40 produces cytotoxicity over an extendedperiod of co-incubation. When this peptide is infused into rat brains,lesions are produced. Some of the proposed breakdown fragments of βAPare also neurotoxic [e.g. βAP (25-35)]. The neurotoxicity appears to beboth mediated via glutamate receptors, and also by non-glutamatereceptors mechanisms. Confocal microscopy studies of neuronal cultureshave demonstrated that exposure to βAP (1-40) results in oxidativestress [Dichlorofluorescein and increased intracellular free calciumFura-2].

In our model it has been demonstrated that βAP fragments can directlyinactivate glutamine synthetase (GS) and creatine kinase (CK) in tissueextracts and in cultured hippocampal neurons and glia (See A and B inFIG. 4). A and B of FIG. 4 present the dose-related inactivation ofglutamine synthetase and creatine kinase by AP (25-35). Cytosolicfractions from gerbil neocortex were prepared and enzyme activitiesdetermined. Samples were incubated in the presence of differentconcentrations of the peptide for 10 min prior to assay. Solid symbolsrepresent the effects of the naturally occurring 25-35 fragment. Opencircles indicate that the reverse sequence (32-25) had no effect onenzyme activity. Open triangles indicate that the scrambled amino acidsequence also had no effect on enzyme activities, compared to the effectof 25-35. Each point is the mean (+/- s.e.) of 5 observations. βAPderived and other cellular sources of free radicals are an importantdeterminant of the initiation and progression of AD.

As demonstrated in C and D in FIG. 4, compound 1 and PBN each show theability to protect GS and CK against the effects of βAP fragments. C andD of FIG. 4 present the protective effects of co-incubation of thecytosolic fractions with BAP 25-35 (0.4 mg/ml) in combination withdifferent concentrations of PBN (open circles) or compound I (closedcircles). Each point is the mean (+/- s.e.) of 3 observations. As can beseen in C and D, compound I gives complete protection and in fact mighteven be able to reverse the effects of oxidation. In contrast, PBN'seffectiveness is quite limited as it is asymptotically leveling out at asubstantially incomplete level of protection.

What is claimed is:
 1. 2,4-disulfonyl α-phenyl tertiary butyl nitrone.2. A compound defined by the formula: ##STR14##
 3. A pharmaceuticallyacceptable salt of ##STR15##
 4. The salt of claim 3 having the formula:##STR16## wherein X is selected from the group consisting of Na, K, NH₄,Ca, Mg, Zn, ZnY, CaY and MgY, wherein Y is a pharmaceutically acceptablemonovalent anion.
 5. A pharmaceutical composition comprising thecompound of claim 1 in a pharmaceutically acceptable intravenousinjectable carrier.
 6. A pharmaceutical composition comprising thecompound of claim 1 in a pharmaceutically acceptable oral carrier.
 7. Apharmaceutical composition comprising the compound of claim 2 in apharmaceutically acceptable intravenous injectable carrier.
 8. Apharmaceutical composition comprising a compound of claim 2 in apharmaceutically acceptable oral carrier.
 9. A pharmaceuticalcomposition comprising the compound of claim 3 in a pharmaceuticallyacceptable intravenous injectable carrier.
 10. A pharmaceuticalcomposition comprising the compound of claim 4 in a pharmaceuticallyacceptable intravenous injectable carrier.
 11. A pharmaceuticalcomposition comprising a compound of claim 3 in a pharmaceuticallyacceptable oral carrier.
 12. A pharmaceutical composition comprising acompound of claim 4 in a pharmaceutically acceptable oral carrier.